PROJECT SUMMARY: Abnormal metabolism is associated with a number of metabolic diseases such as cancer, diabetes, and cardiovascular diseases. Important markers for the altered metabolic states include changes in fuel preferences and loss of acidity regulations in tissues. To date, routine assessments of energy metabolism and acidity in viable tissues remain a challenge. Sensitive imaging modalities that can provide accurate characterization of these important biomarkers are therefore highly desirable. In this application, we aim to establish a series of hyperpolarized (HP) 13C-enriched molecules that can be activated by an endogenous enzyme esterase to produce both pH and metabolic imaging probes in tissues. Successful development of these HP 13C-enriched compounds will allow for tissue acidity and metabolic information to be simultaneously characterized following an injection of a single imaging probe. Molecular candidates for these applications must be chemically stable, can be highly polarized by dynamic nuclear polarization (DNP), have long 13C T1?s, and be highly sensitive to esterase hydrolysis. Once hydrolyzed, the molecules must quickly decompose to form a pair of pH imaging probes, HP 13CO2 and HP H13CO3-, as well as a HP 13C-enriched metabolic substrate such as 13C- pyruvate. Here, we propose a series of ethyl alkyl mixed anhydride carbonate compounds with 13C- enrichments at both the carbonate and carboxyl carbons. These carbon centers have long T1 making them suitable for HP 13C imaging applications. Moreover, these small organic molecules are expected to be efficiently polarized by DNP with significantly improved 13C MR imaging sensitivity. Last but not least, the ester functional group in these mixed anhydrides is expected to be highly susceptible to hydrolysis by esterase. Once injected into the circulation, we expect that these HP 13C-mixed anhydride carbonates will be rapidly hydrolyzed by esterase producing monoacyl carbonate molecules. These intermediates are expected to decompose, producing HP 13CO2 and a HP 13C-enriched metabolic substrate for pH measurement and metabolism analysis in tissues, respectively. In Aim 1 of this study, we will synthesize 13C-enriched ethyl acyl mixed anhydride carbonates with 13C-acetate, 13C-pyruvate, or 13C-butyrate as the acyl moiety. HP 13C parameters such as T1, signal enhancement and chemical stability of these compounds will also be evaluated. In Aim 2, we will investigate the production of HP 13CO2/H13CO3- and HP 13C metabolic substrate by esterase hydrolysis in solutions of isolated esterase, isolated rat plasma, blood, and homogenates of rat liver and heart tissues. Finally, we will evaluate the acute toxicity as well as the in vivo imaging efficacy of these HP 13C probes in mice and rats (Aim 3). Successful execution of this study could lead to a series of HP 13C imaging probes that allow for simultaneous imaging of tissue pH and energy metabolism in tissues associated with metabolic diseases.